细菌整体代谢与新抗生素及抗生素增效剂靶标

摘要：抗菌药物的滥用加速了细菌耐药的产生与传播,每年因耐药细菌导致的人口死亡和医疗成本耗费都极为惊人。针对 耐药细菌的新型抗生素研制十分缓慢,自1987年以来没有一类新型抗生素上市。研究抗生素杀菌机制以开发新药或抗生素佐剂 是一种应对耐药细菌的良好策略。目前普遍认同活性氧(ROS)介导细胞死亡是抗生素杀菌的共享途径,抗生素作用于靶标介导 产生原发初级损伤,诱使ROS生成,ROS造成次级细胞损伤并刺激更多ROS生成,形成一种恶性循环,最终ROS累积超过细胞 氧化应激极限、致使细菌死亡。细菌胞内具有用以消除氧化应激压力的专能系统,针对性抑制这些系统可能是快速杀伤细菌的 一个好策略。然而,设计靶向细菌某些代谢节点的新型抗生素或相关佐剂很可能更为简单、有效,生物体代谢是一个巨大的相 互协作网络,关键节点的扰动很容易引起代谢通路的剧烈波动,能引发细菌整体氧化应激状态改变的代谢节点有望成为潜在新 抗生素或抗生素增效剂的靶标。     

氨基糖苷类抗生素作用机制研究进展

氨基糖苷类抗生素作为一种杀菌性抗菌药物,具有抗菌谱广、杀菌完全等特点,其作用机制主要是通过抑制细菌蛋白质的合成而杀死细菌。随着新技术、新方法的应用,杀菌性抗生素普遍性地通过产生羟基自由基杀死细菌及其他作用途径也被挖掘。本文简要综述氨基糖苷类抗生素的作用机制,并对新型氨基糖苷类抗生素的开发进行展望。     

活性氧在细菌耐抗生素机制中的作用

细菌对抗生素的耐药性以惊人的速度蔓延,阐明抗生素导致细菌死亡内在机制,对提高抗生素药效以及寻找新型抗生素显得尤为迫切。近期研究表明,抗生素引起的细菌内活性氧物质(reactive oxygen species,ROS)产生,是导致细菌细胞死亡的关键原因。该文对ROS、SOS和细菌耐药性方面的最新研究进行了综述和探讨,为开发新型抗菌药物,遏制细菌感染和耐药性提供新的思路。     

抗生素耐药性的主动外排机制

细菌对抗生素产生耐药性的机制多种多样，常见的有：细菌产生酶对抗生素的修饰与灭活；药物作用靶点发生改变；膜通透性下降；膜产生主动外排作用；生物被膜的形成。膜的主动外排机制是由各种外排蛋白系统介导的把抗生素等药物从细菌细胞内泵出的主动排出过程，外排系统具有能量依赖性、底物广泛性、系统多样性及功能复杂性的特点；与常用抗生素、合成抗菌药、染料、去污剂及金属离子的多重耐药性密切相关，尤其对四环素、氯霉素及氟喹诺酮类药物等广谱抗菌药的作用更为明显；其生理功能与正常物质转运和代谢有关。以利血平为代表的外排泵抑制剂能有效地对抗主动外排作用的产生，此类抑制剂将是防制细菌多重耐药性的有效措施之一。     

细菌耐药性监测分析中应注意的问题

细菌耐药性监测对于了解本地区细菌耐药性现状和发展趋势、指导临床合理使用抗生索具有重要意义。为此，临床微生物学工作者应认真掌握临床常见细菌的某些特性及抗生素的相关知识，如天然耐药性、罕见耐药谱型、易产生选择性耐药的抗生素及代表性药物在药敏试验中的作用。因为这些知识对于如何解释药敏试验结果和监测数据分析时至关重要。     

抗生素杀菌机制与Fenton反应有关

抗生素作用机制包括抑制细胞壁和生物大分子(如DNA、RNA、蛋白质和叶酸)合成,但抑制过程如何导致细菌死亡仍是一个谜。Cell上一篇论文报道:不同杀菌机制的抗生素杀死细菌时表现出一种相同机制———生成羟基。抗生素分为杀菌型和抑菌型两类。Collins等曾发现用喹诺酮类对大肠埃     

抗生素药性的主动外排机制

细菌对抗生素产生耐药性的机制多种多样，常见的有；细菌产生酶对抗生素的修饰与灭活；药物作用靶点发生改变；膜通透性下降；膜产生主动外排作用；生物被膜的形成，膜的主动外排机制是由各种外排蛋白系统介导的把抗生素等药物从细菌细胞内泵出的主动排出过程，外排系统具有能量依赖性，底物广泛性，系统多样性及功能复杂性的特点；与常用抗生素，合成抗菌药，染料，去污剂及金属离子的多重耐药性密切相关，尤其对四环素，氯霉素及氟喹诺酮类药等广谱抗菌药的作用更为明显；其生理功能与正常物质转运和代谢有关，以利血平为代表的外排泵抑制剂能有效地对抗主动外排作用的产生，此类抑制剂将是防制细菌多重耐药性的有效措施之一。     

多药耐药流出泵的作用

过去几十年里，细菌对许多抗生素都产生了耐药性，甚至一些抗生素已丧失抗感染疗效，而且随着其种类的增多，细菌可对多种类型的抗生素产生耐药性。细菌的耐药机制主要包括：阻止抗生素进入细菌细胞中；通过流出泵把抗生素泵出细菌体外，即抗生素的流出。一些抗菌药不能用来抗多种革兰阴性菌引起的感染，因为这些细菌对前者有内在耐药作用。最初认为这种作用是由细菌外膜对药物的低渗透性引起的，后经研究发现，绿脓杆菌对几种抗生素的内在耐药是由流出作用引起的，一些药物因受流出作用影响而对革兰阴性菌感染收效甚微。细菌对人用和兽用抗生素产生的耐药性，可能是流出泵生理作用的副产物。     

酸奶能拯救抗生素横扫过的肠胃吗？

服用抗生素后，你出现了食欲不振、便秘、腹泻等等胃肠不适的状况？那就需要补充益生菌，来恢复你肠道菌群的生态平衡了。 抗生素的主要作用是杀菌。对于细菌，它宁可错杀一万，也不放过一个。尤其是广谱抗生素（如头孢曲松、美洛培南等），具有更为广泛和强大的杀菌功能，益生菌更是不可避免地成了被消灭的对象。当正常菌群中的敏感细菌受到抑制或杀灭时，耐药菌（如葡萄球菌等）就会趁机大量繁殖，就会造成肠道细菌群的紊乱，出现食欲不振，免疫力下降等症状。     

滥用抗生素在临床造成不良反应的分析

抗生素是指微生物在生长过程中，为了生存竞争的需要产生抑制或杀灭其他微生物的化学物质。自抗生素问世以来，在感染性疾病的治疗上发挥着重要作用。抗生素是主要用于治疗细菌性感染的药物，但它并不是万能的，不能包治百病。抗生素只有杀灭或抑制细菌的作用，对病毒无效。一般来讲，在没有合并感染的情况下不必应用抗生素。由过敏、冻伤等引起的炎性反应及真菌感染原则上也不宜用抗生素。随着抗生素的广泛应用，产生了细菌的多重耐药和致病力增强，造成二重感染和医院内感染的大量增加。     

Mouse Melanoma Cell Migration is Dependent on Production of Reactive Oxygen Species under Normoxia Condition

Cell migration plays a role in many physiological and pathological processes. Reactive oxygen species (ROS) produced in mammalian cells influence intracellular signaling processes which in turn regulate various biological activities. Here, we investigated whether melanoma cell migration could be controlled by ROS production under normoxia condition. Cell migration was measured by wound healing assay after scratching confluent monolayer of B16F10 mouse melanoma cells. Cell migration was enhanced over 12 h after scratching cells. In addition, we found that ROS production was increased by scratching cells. ERK phosphorylation was also increased by scratching cells but it was decreased by the treatment with ROS scavengers, N-acetylcysteine (NAC). Tumor cell migration was inhibited by the treatment with PD98059, ERK inhibitor, NAC or DPI, well-known ROS scavengers. Tumor cell growth as judged by succinate dehydrogenase activity was inhibited by NAC treatment. When mice were intraperitoneally administered with NAC, the intracellular ROS production was reduced in peripheral blood mononuclear cells. In addition, B16F10 tumor growth was significantly inhibited by in vivo treatment with NAC. Collectively, these findings suggest that tumor cell migration and growth could be controlled by ROS production and its downstream signaling pathways, in vitro and in vivo.     

Toll-like receptor (TLR) response tolerance: a key physiological "damage limitation" effect and an important potential opportunity for therapy

Endotoxin tolerance is a well known phenomenon, described both in vivo and in vitro, in which repeated exposure to endotoxin results in a diminished response, usually characterised as a reduction in pro-inflammatory cytokine release. The mechanisms responsible for endotoxin tolerance have become clear in recent years as our understanding of the pathways through which endotoxin mediates its effects has increased. The principal cell surface receptor for the lipopolysaccharide (LPS) component of endotoxin is Toll-Like Receptor 4 (TLR-4), a member of a highly conserved family of receptors specific for highly conserved bacterial and viral components which play key roles in the early inflammatory response to pathogens. As our understanding of the part played by TLR-4 signalling in endotoxin has increased, so it has become clear that response tolerance occurs to other TLR ligands in addition to LPS/endotoxin. Clinically, endotoxin/TLR response tolerance is thought to play an important part in susceptibility to reinfection in patients treated for severe sepsis. Whilst this may have developed as a protective evolutionary mechanism to prevent death caused by overwhelming cytokine release in sepsis, in the modern era of antibiotics, vasopressors and organ support, undoing this downregulation or "re-booting" the immune system may be a useful therapeutic target in the post-septic patient. This should, however, be approached with caution as it is possible that endotoxin/TLR response tolerance is also a physiological regulatory mechanism in areas normally exposed to bacterial-derived TLR-ligands such as the gut and liver.     

Targeting antioxidants for cancer therapy

Cancer cells are characterized by an increase in the rate of reactive oxygen species (ROS) production and an altered redox environment compared to normal cells. Furthermore, redox regulation and redox signaling play a key role in tumorigenesis and in the response to cancer therapeutics. ROS have contradictory roles in tumorigenesis, which has important implications for the development of potential anticancer therapies that aim to modulate cellular redox levels. ROS play a causal role in tumor development and progression by inducing DNA mutations, genomic instability, and aberrant pro-tumorigenic signaling. On the other hand, high levels of ROS can also be toxic to cancer cells and can potentially induce cell death. To balance the state of oxidative stress, cancer cells increase their antioxidant capacity, which strongly suggests that high ROS levels have the potential to actually block tumorigenesis. This fact makes pro-oxidant cancer therapy an interesting area of study. In this review, we discuss the controversial role of ROS in tumorigenesis and especially elaborate on the advantages of targeting ROS scavengers, hence the antioxidant capacity of cancer cells, and how this can be utilized for cancer therapeutics.     

Effects of sub-minimum inhibitory concentrations of ciprofloxacin on enteroaggregative Escherichia coli and the role of the surface protein dispersin

Enteroaggregative Escherichia coli (EAEC) are bacterial pathogens that cause watery diarrhoea, which is often persistent and can be inflammatory. The antibiotic ciprofloxacin is used to treat EAEC infections, but a full understanding of the antimicrobial effects of ciprofloxacin is needed for more efficient treatment of bacterial infections. In this study, it was found that sub-minimum inhibitory concentrations (sub-MICs) of ciprofloxacin had an inhibitory effect on EAEC adhesion to glass and mammalian HEp-2 cells. It was also observed that bacterial surface properties play an important role in bacterial sensitivity to ciprofloxacin. In an EAEC mutant strain where the hydrophobic positively charged surface protein dispersin was absent, sensitivity to ciprofloxacin was reduced compared with the wild-type strain. Identified here are several antimicrobial effects of ciprofloxacin at sub-MIC concentrations indicating that bacterial surface hydrophobicity affects the response to ciprofloxacin. Investigating the effects of sub-MIC doses of antibiotics on targeted bacteria could help to further our understanding of bacterial pathogenicity and elucidate future antibiotic treatment modalities.     

Antioxidant strategies for neurodegenerative diseases

A very large body of evidence supports the involvement of oxidative stress in neurodegenerative pathologies. Therefore, therapies based on administration of antioxidant and reactive oxygen species (ROS) scavenging substances have been widely proposed for treatment of both acute and chronic neuropathological diseases. In the present review, several recent patents (mostly from 1998 - 2000) based on the proposed use of antioxidant strategies to treat neurodegenerative diseases are considered. In particular, the following classes of patents are examined: patents for novel antioxidant and ROS scavengers, pharmaceutical compositions in which antioxidant and ROS scavengers are claimed together with other pharmacological tools, patents based on new biotechnological and gene therapy approaches, and patents based on enzymatic manipulation of ROS production and elimination. In the comment, the most promising perspectives stemming from these patents are discussed; in particular: (i.) the usefulness of pharmaceutical compositions able to counteract the deleterious effects of excessive amounts of free radicals originating from different cellular process, without blocking the physiological beneficial action of their controlled production; (ii.) the potential beneficial effects of combining antioxidant treatments with modulation of the activity of receptors for excitatory amino acid and modulation of calcium levels; (iii.) the tremendous future impact of biotechnological and gene therapy techniques.     

Effects of copper and temperature on aquatic bacterial communities

The present study aimed to characterise effects of copper and temperature on bacterial communities in photosynthetic biofilms using a suit of supplementary methods: pollution-induced community tolerance (PICT), DNA profiles with denaturing gradient gel electrophoresis (DGGE) and physiological profiles with community-level physiological profiling (CLPP). Biofilms of algae and bacteria were grown in a ditch of a Dutch polder and exposed in the laboratory to copper (3 microM and a reference) at three different temperatures (10, 14 and 20 degrees C). Bacterial communities sampled from the field showed heterogeneity in their physiological profiles, however the heterogeneity decreased during laboratory incubation. After 3 days laboratory incubation, the copper treated biofilms were different from the reference biofilms, as revealed by DGGE and CLPP analyses. Effects of temperature were not observed in the CLPPs, or in the DGGE profiles. PICT was observed for the bacterial communities at all temperatures. The copper-tolerance at 10 and 14 degrees C increased about 3 times, whereas copper-tolerance at 20 degrees C increased about 6 times. Temperature had an effect on the community tolerance, but not on the structure or on the physiological profile, suggesting that temperature was not a major factor causing successional changes under these laboratory conditions. In contrast, temperature had an effect on tolerance development indicating that the exposure to copper was enhanced at higher temperature.     

Role of engineered metal oxide nanoparticle agglomeration in reactive oxygen species generation and cathepsin B release in NLRP3 inflammasome activation and pulmonary toxicity

Incomplete understanding of the contributions of dispersants and engineered nanoparticles/materials (ENM) agglomeration state to biological outcomes presents an obstacle for toxicological studies. Although reactive oxygen species (ROS) production is often regarded as the primary indicator of ENM bioactivity and toxicity, it remains unclear whether ENM produce ROS or whether ROS is an outcome of ENM-induced cell injury. Phagolysosomal disruption and cathepsin B release also promote bioactivity through inflammasome activation. Therefore, specific particle parameters, i.e. preexposure dispersion status and particle surface area, of two ENM (NiO and CeO₂) were used to evaluate the role of ROS generation and cathepsin B release during ENM-induced toxicity. Male C57BL/6J mice were exposed to 0, 20, 40, or 80?μg of poorly or well-dispersed NiO-NP or CeO₂-NP in four types of dispersion media. At 1- and 7-day postexposure, lung lavage fluid was collected to assess inflammation, cytotoxicity, and inflammasome activation. Results showed that preexposure dispersion status correlated with postexposure pulmonary bioactivity. The differences in bioactivity of NiO-NP and CeO₂-NP are likely due to NiO-NP facilitating the release of cathepsin B and in turn inflammasome activation generating proinflammatory cytokines. Further, both metal oxides acted as free radical scavengers. Depending on the pH, CeO₂-NP acted as a free radical scavenger in an acidic environment (an environment mimicking the lysosome) while the NiO-NP acted as a scavenger in a physiological pH (an environment that mimics the cytosol of the cell). Therefore, results from this study suggest that ENM-induced ROS is not likely a mechanism of inflammasome activation.     

Gangliosides induce autophagic cell death in astrocytes

Background and purpose:

Gangliosides, sialic acid-containing glycosphingolipids, abundant in brain, are involved in neuronal function and disease, but the precise molecular mechanisms underlying their physiological or pathological activities are poorly understood. In this study, the pathological role of gangliosides in the extracellular milieu with respect to glial cell death and lipid raft/membrane disruption was investigated.

Experimental approach:

We determined the effect of gangliosides on astrocyte death or survival using primary astrocyte cultures and astrocytoma/glioma cell lines as a model. Signalling pathways of ganglioside-induced autophagic cell death of astrocytes were examined using pharmacological inhibitors and biochemical and genetic assays.

Key results:

Gangliosides induced autophagic cell death in based on the following observations. Incubation of the cells with a mixture of gangliosides increased a punctate distribution of fluorescently labelled microtubule-associated protein 1 light chain 3 (GFP-LC3), the ratio of LC3-II/LC3-I and LC3 flux. Gangliosides also increased the formation of autophagic vacuoles as revealed by monodansylcadaverine staining. Ganglioside-induced cell death was inhibited by either a knockdown of beclin-1/Atg-6 or Atg-7 gene expression or by 3-methyladenine, an inhibitor of autophagy. Reactive oxygen species (ROS) were involved in ganglioside-induced autophagic cell death of astrocytes, because gangliosides induced ROS production and ROS scavengers decreased autophagic cell death. In addition, lipid rafts played an important role in ganglioside-induced astrocyte death.

Conclusions and implications:

Gangliosides released under pathological conditions may induce autophagic cell death of astrocytes, identifying a neuropathological role for gangliosides.     

DNA damage caused by metal nanoparticles: involvement of oxidative stress and activation of ATM

Nanotechnology is a fast growing emerging field, the benefits of which are widely publicized. Our current knowledge of the health effects of metal nanoparticles such as nanosized cobalt (Nano-Co) and titanium dioxide (Nano-TiO(2)) is limited but suggests that metal nanoparticles may exert more adverse pulmonary effects as compared with standard-sized particles. To investigate metal nanoparticle-induced genotoxic effects and the potential underlying mechanisms, human lung epithelial A549 cells were exposed to Nano-Co and Nano-TiO(2). Our results showed that exposure of A549 cells to Nano-Co caused reactive oxygen species (ROS) generation that was abolished by pretreatment of cells with ROS inhibitors or scavengers, such as catalase and N-acetyl-L(+)-cysteine (NAC). However, exposure of A549 cells to Nano-TiO(2) did not cause ROS generation. Nano-Co caused DNA damage in A549 cells, which was reflected by an increase in length, width, and DNA content of the comet tail by the Comet assay. Exposure of A549 cells to Nano-Co also caused a dose- and a time-response increased expression of phosphorylated histone H2AX (γ-H2AX), Rad51, and phosphorylated p53. These effects were significantly attenuated when A549 cells were pretreated with catalase or NAC. Nano-TiO(2) did not show these effects. These results suggest that oxidative stress may be involved in Nano-Co-induced DNA damage. To further investigate the pathways involved in the Nano-Co-induced DNA damage, we measured the phosphorylation of ataxia telangiectasia mutant (ATM). Our results showed that phosphorylation of ATM was increased when A549 cells were exposed to Nano-Co, and this effect was attenuated when cells were pretreated with catalase or NAC. Pretreatment of A549 cells with an ATM specific inhibitor, KU55933, significantly abolished Nano-Co-induced DNA damage. Furthermore, pretreatment of A549 cells with ROS scavengers, such as catalase and NAC, significantly abolished Nano-Co-induced increased expression of phosphorylated ATM. Taken together, oxidative stress and ATM activation are involved in Nano-Co-induced DNA damage. These findings have important implications for understanding the potential health effects of metal nanoparticle exposure.     

Bacterial translocation, microcirculation injury and sepsis

Sepsis is the result from a complex bacterial-host interaction, which is an often-fatal response when host protective molecular mechanisms designed to fight invading bacteria surpass the beneficial intensity to the point of causing injury to the host. Increasing evidences have implicated the bacterial translocation (BT) as the main source for the induction of sepsis, although the beneficial effect of BT process has been related to the development of the intestinal immune response by physiological interaction between bacteria and host. In this article, we examined evolving concepts concerning to BT and discussed about its potential role in the promotion of microcirculation injury, moreover, its possible participation in the sepsis induction. According to our data obtained from in-vivo BT animal-model, both bacterial overgrowth and bacterial pathogenic determinants seem to be major predisposing factors for the induction of BT. Besides, translocation of luminal bacteria through the lymphatic via elicits the activation of the GALT inflammatory response contributing to microcirculation injuries, and the haematological via of BT was responsible to the systemic bacterial spread. On other hand, the combination of BT process to the pre-existing host systemic infection played a crucial role in the worsening of the clinical outcome. In our understanding, studies concerning to intestinal immune response and the pathophysiology of bacterial-host interaction, under normal and disease conditions, seems to be the key elements to the development of therapeutic approaches towards sepsis.